(1) Summary of the Invention
The present invention relates to a method for quantitatively chemically profiling mammalian cells involved in a disease using capillary electrophoresis. The present invention is particularly effective in testing for metabolites of cells in diabetes. The method can be used to test the effectiveness of aldose reductase inhibitors.
(2) Description of Related Art
Capillary electrophoresis is well known to those skilled in the art. Illustrative are U.S. Pat. No. 5,213,669 to Guttman; U.S. Pat. No. 5,370,777 to Guttman et al; U.S. Pat. No. 5,431,793 to Wang et al; U.S. Pat. No. 5,490,909 to Wang et al; PCT 93/15395 and Jellum, E., et al., J. of Chromatography 559:455-465 (1991). The method has been used with proteins, amino acids and nucleotides.
Presently, great interest exists in the changes of metabolites in cells exposed to elevations in glucose as in diabetes. These models include: tissue culture models of diabetes, transgenic diabetic models, animal models of diabetes, human models including clinical intervention trials to ameliorate the effects of diabetes on tissues (nerve, kidney, retinal and blood vessels) involved in the long-term complications from the disease. In order to study changes of intracellular metabolites in diabetes, investigators have had to use high performance liquid chromatography (HPLC) or bioassays. These assays are laborious, expensive, time consuming, often frustrated by technical problems and require large amounts of tissue to examine. Also, HPLC or bioassays require hours to days to run and standardize data. By example, Phase III clinical trials on the efficacy of aldose reductase inhibitors in the treatment and/or prevention of long-term complications (neuropathy) from diabetes are presently underway. These human trials require the quantitation of sorbitol and myo-inositol from peripheral nerve biopsy (such as the sural nerve). The specimen of nerve tissue must undergo derivitization and extraction of carbohydrates for the determination of tissue sorbitol and myo-inositol levels by HPLC which necessitates the use of almost one cm of nerve biopsy. There is a need for a faster and better method for analyzing for changes in the metabolites.
While the findings of the Diabetes Control and Complications Trial have incontrovertibly removed any doubt regarding the role of elevated blood glucose and the risk for long-term complications from diabetes, the mechanism(s) of tissue injury in diabetes are poorly understood (The DCCT Research Group. N. Engl J Med (September 30) 329 (14):977-986 (1993)). Interrelated metabolic imbalances linked to diabetes and glucose induced cellular dysfunction have been proposed as putative pathogenic mechanisms resulting in the vascular, renal, retinal and nerve complications of diabetes. These interrelated metabolic pathways (FIG. 1) include increased flux or transport of glucose into the cell (Henry, D. N., et al., J. Am Society of Nephr. (7)9:1871 (1996): and Heilig, C. W., et al., J. Clin. Invest. 96:1802-1814 (1995)), polyol metabolism with the accumulation of sorbitol and change in myo-inositol (MI) concentration (Gabbay, K. H., Annu Rev Med:26:521-536 (1975); and Winegrad, A. I., et al., N Engl J Med 16:295(25):1416-1421 (Dec. 16, 1976)), altered cellular redox potential (Tilton, R. G., et al., Kidney International 41:778-788 (1992); and Williamson, J. R., et al., Diabetes 42:801-813 (1993)), de novo formation of diacylglycerol (DAG) leading to activation of protein kinases (PKC) (Wolf, B. A., et al., J Clin Invest 87:31-38 (1990)), decreased nitric oxide (endothelial derived relaxation factor) formation (Greene, D. A., et al., Diabetes and Metabolism Reviews 9(3):189-217 (1993); and Stevens, J. J., Diabetic Medicine 12:292-295 (1995)), and non-enzymatic glycosylation of proteins (Brownlee, M., et al., Ann Intern Med Oct; 101(4):527-537 (1984)). Presently, there are no means to concomitantly measure the metabolic intermediates and/or end-products of these pathways as they change in diabetes.
CE has been applied to the diagnosis and studies of human disease, particularly metabolic disorders such as homocytinuia, cystinuria, glutationone synthetase and adenylsuccinases deficiencies in urine and blood (Jellum, E., et al., J. Chromatography 559:455-465 (1991)). CE has also been successfully employed to measure nitrite and nitrates in plasma as indirect measures of nitric oxide in human subjects (Leone, A. M., et al., Biochem Biophy Res Comm 200 (2):951-957 (1994); and Ueda, T., et al., Electrophoresis 16:1002-1004 (1995)). CE has been used to test for glycosolated hemoglobin determination in diabetes (Doelman, C. J., et al., Clin Chem Apr 43 (4):644-648 (1997); and Lillard, S. J., et al., J Chromatogr B Biomed Appl Dec 13; 687 (2):363-369 (1996)). The analysis of single cells for protein is described by Lee et al, Anal. Chem 64, 3045-3051 (1992). What is needed is a method which provides a complete profile of the components of multiple cells from the same sample. This provides a basis for determining whether or not the cells are diseased.
It is an object of the present invention to chemically profile mammalian cells in vitro in the presence of or absence of compounds which affect the profile using capillary electrophoresis. In particular, it is an object of the present invention to provide an in vitro method which uses capillary electrophoresis to measure intracellular metabolites, proteins and signal transduction molecules from tissues and cultures exposed to disease conditions on cellular metabolism in vitro and in vivo. Further, it is an object of the present invention to provide a method which is much simpler and more economical than high pressure chromatography (HPLC). These and other objects of the present invention will become increasingly apparent by reference to the following description and the drawings.